Inhibition of TNF activity

ABSTRACT

The bioactivity of TNF is inhibited by administering heparin or a derivative thereof along with a soluble TNF receptor. The heparin or derivative thereof can be administered simultaneously with the soluble TNF receptor, either in separate compositions or in compositions containing both heparin or a derivative thereof and at least one soluble TNF receptor. The heparin or derivative may also be administered without the soluble TNF receptor and still effect some amount of inhibition of TNF bioactivity.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL99/00709, filed Dec. 30, 1999.

FIELD OF THE INVENTION

The present invention is directed to a method and pharmaceuticalcompositions for inhibiting activity of tumor necrosis factor (TNF).

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine produced by awide spectrum of cells. It has a key role in defending the host,mediating complex cellular responses of different, and even contrasting,nature (Aggarwal et al, 1996). In excess, TNF may have detrimentalsystemic effects. Two specific high affinity cell surface receptors, thep55 TNF-receptor (p55 TNF-R) and the p75 TNF-receptor (p75 TNF-R),function as transducing elements, providing the intracellular signal forcell responses to TNF. The extracellular parts of the TNF-Rs, known assoluble TNF-Rs, were formerly referred to as TBP-I and TBP-IIrespectively (see Wallach, U.S. Pat. No. 5,359,037 and Tartaglia et al.,1992; Loetscher et al., 1991).

The biological effects of TNF depend upon its concentration and site ofproduction. At low concentrations. TNF may produce desirable homeostaticand defense functions. For example, these effects may destroy tumorcells or virus infected cells and augment antibacterial activities ofgranulocytes. In this way, TNF contributes to the defense of theorganism against infectious agents and to recovery from injury. However,at higher concentrations, systemically or in certain tissues, TNF cansynergize with other cytokines, notably interleukin-1, to aggravate manyinflammatory responses. Additionally, the effects of TNF-α, primarily onthe vasculature, are now known to be a major cause for symptoms ofseptic shock (Tracey et al, 1986). In some diseases, TNF may causeexcessive loss of weight (cachexia) by suppressing activities ofadipocytes and by causing anorexia.

TNF has been found to induce the following activities (together withinterleukin-2): fever, slow-wave sleep, hemodynamic shock, increasedproduction of acute phase protein, decreased production of albumin,activation of vascular endothelial cells, increased expression of majorhistocompatibility complex molecules, decreased lipoprotein lipase,decreased cytochrome P450, decreased plasma zinc and iron, fibroblastproliferation, increased synovial cell collagenase, increasedcyclo-oxygenase activity, activation of T cells and B cells, andinduction of secretion of the cytokines, TNF itself, interleukin-1 andinterleukin-6.

Because of its pleiotropic effects, TNF has been implicated in a varietyof pathologic states in many different organs of the body. In bloodvessels, TNF promotes hemorrhagic shock, down-regulates endothelial cellthrombomodulin, and enhances a procoagulant activity. It causes adhesionof white blood cells, and probably of platelets, to the walls of bloodvessels, and so may promote processes leading to atherosclerosis, aswell as to vasculitis.

TNF activates blood cells and causes the adhesion of neutrophils,eosinophils, monocytes/macrophages and T and B lymphocytes. By inducinginterleukin-6 and interleukin-8, TNF augments the chemotaxis ofinflammatory cells and their penetration into tissues. Thus, TNF has arole in the tissue damage of autoimmune disease, allergies and graftrejection.

TNF has also been called cachectin because it modulates the metabolicactivities of adipocytes and contributes to the wasting and cachexiaaccompanying cancer, chronic infections, chronic heart failure andchronic inflammation. TNF may also have a role in tissue damage ofautoimmune diseases, allergies, and graft rejection.

TNF also has metabolic effects on skeletal and cardiac muscle. It alsohas marked effects on the liver: it depresses albumin and cytochromeP450 metabolism and increases production of fibrinogen, α-AcidGlycoprotein (AGP) and other acute phase proteins. It can also causenecrosis of the bowel.

In the central nervous system, TNF crosses the blood-brain barrier andinduces fever, increased sleep and anorexia. Increased TNF concentrationis also associated with multiple sclerosis. It also causes adrenalhemorrhage and affects production of steroid hormones, enhancescollagenase and PGE-2 in the skin, and causes the breakdown of bone andcartilage by activating osteoclasts.

Thus, TNF is involved in the pathogenesis of many undesirableinflammatory conditions, in autoimmune disease, graft, rejection,vasculitis and atherosclerosis. It appears to have a role in heartfailure, in the response to cancer and in anorexia nervosa. For thesereasons, means have been sought to inhibit the activity of TNF as a wayto control a variety of diseases.

While exploring ways for antagonizing the destructive potential of TNFin certain clinical conditions, investigators looked for natural TNFinhibitors (Engelmann et al, 1989; Engelmann et al, 1990; Seckinger etal, 1989; Olsson et al, 1989). Such agents, first detected in urine,were structurally identical to the extracellular cytokine bindingdomains of the two membrane associated TNF-Rs (Nophar et al, 1990).These shed soluble TNF-Rs (sTNF-Rs) can compete for TNF with the cellsurface receptors and thus block the cytokine activity.

However, interactions between the TNF-Rs and their ligand are much morecomplex than initially thought. At physiological concentrations, thetrimeric and bioactive TNF molecules decay, dissociating into inactivemonomeric forms (Petersen et al, 1989; Aderka et al, 1992). Addition ofsTNF-Rs to the TNF trimers promotes formation of complexes between them,which can preserve and prevent the decay of the active, trimeric formsof TNF (Aderka et al, 1991; De Groote et al, 1993). This bioactive TNFmay dissociate from this complex to replace free TNF which decayed, thusmaintaining a constant concentration of free, bioactive, trimericcytokine. This reversible interaction between the soluble receptors andtheir ligand expands the functions attributable to the TNF receptors. Intheir soluble form, the TNF-Rs may serve as:

(a) TNF antagonists (when present in large excess relative to TNF);

(b) TNF carrier proteins (between body compartments);

(c) slow release reservoirs for bioactive TNF;

(d) stabilizers of the TNF's bioactive form (which may also prolong thehalf-life of TNF); and

(e) TNF “buffers”, by inhibiting the effects of high TNF concentrationsand presenting it at low and well-controlled levels to the cells (Aderkaet al, 1992).

The functions of the TNF receptors, thus, are not limited to signaltransduction but include, in their soluble forms, extracellularregulatory roles affecting local and systemic bioactive TNFavailability.

TNF and Disease

Examination of patients with septic shock due to meningococcemiarevealed that the ratio of TNF/sTNF-Rs was higher in patients with afatal outcome compared to patients who recovered, suggesting a criticalimbalance between the ligand and its inhibitors (Girardin et al, 1994).Neutralization of the excess TNF seemed to be the preferred next step.

Indeed, dimeric Fc fusion constructs of the p55 sTNF-R, but not of thep75 sTNF-R, were found to protect mice from lethal doses of LPS (Evanset al, 1994) if administered not later than 1-3 hours post LPS (Peppelet al, 1991; Mohler et al, 1993; Ashkenazi et al, 1991; Lesslauer et al,1991). This suggests that septic shock manifestations occur if theinitial high TNF concentrations generated are not buffered by adequatesoluble receptor concentrations during that narrow window of time.

To add to the growing confusion, neutralization of TNF with monoclonalanti-TNF Ab (Abraham et al, 1995; Kaul, et al, 1996) or p55 sTNF-R IgG1(Leighton et al, 1996) in patients with severe sepsis or septic shockyielded conflicting results. In one study, the antibodies provedineffective (Abraham et al, 1995), while in the other, administration ofthe antibodies benefited only those patients with baseline interleukin-6levels higher than 1000 pg/ml but increased the mortality of those withlower interleukin-6 levels (Kaul et al, 1995). In another randomizedtrial, septic patients given a recombinant dimer consisting of sTNF-R/Fcportion of IgG1 had higher mortality (48-53%) as compared toplacebo-treated patients (30%) (Suffredini et al, 1994; Fisher et al,1996). Interestingly, the higher the dose of the sTNF-Rs administered,the higher was the patient mortality (Fisher et al, 1996). It wassuspected that the effective removal of circulating TNF may result inthe exacerbation of the systemic infection (Fisher et al, 1996). Incontrast, in a recent study the administration of similar soluble Fcreceptor constructs apparently benefitted septic patients irrespectiveof their serum interleukin-6 concentrations, with a 36% mortalityreduction compared to placebo treated individuals (Leighton et al,1996). These contradictory data give the impression that theadministration of sTNF-Rs may have a very narrow therapeutic index whichwould be difficult to individualize at bedside. Too much of thereceptors may totally neutralize TNF, exacerbating the systemicinfection, while too little of the receptors may not neutralize enoughTNF, resulting in septic shock and the patient's demise. The realchallenge is to fine-tune the sTNF-R dose in order to permit low TNFlevels to exert their protective effects. Thus, paradoxically, lowerdoses of sTNF-Rs than previously employed (Fisher et al, 1996), ratherthan higher ones, may benefit septic shock patients.

Since the TNF neutralization should not be complete, but should be aimedto leave low amounts of bioactive TNF to exert the desired beneficialeffects, natural soluble TNF receptors may be ideally suited for thispurpose.

TNF is also a pivotal cytokine in the pathogenesis of Crohn's Disease, achronic and disabling disorder of the bowel, and is, therefore, a primetarget for specific immunotherapy (Braegger et al, 1992; MacDonald etal, 1990; Breese et al, 1994). Indeed, treatment of Crohn's Diseasepatients with chimeric anti-TNF monoclonal antibodies induced aspectacular remission in patients unresponsive to conventional therapy(van Dullemen et al, 1995). Whether slow release preparations of sTNF-Rs(Eliaz et al, 1966) will have identical effects on the course of thisdisease remains to be determined.

In another autoimmune disorder, rheumatoid arthritis, it wasdemonstrated that the serum sTNF-Rs may be useful in monitoring diseaseactivity (Cope et al, 1992; Roux-Lombard et al, 1993). It was shown thatdespite the presence of high levels of TNF inhibitors in joints affectedby rheumatoid arthritis, these inhibitors were insufficient toneutralize TNF activity (Cope et al, 1992). A randomized double blindstudy comparing administration of chimeric anti-TNF monoclonalantibodies to patients with rheumatoid arthritis resulted in animpressive clinical remission (Levine et al, 1994). Recently, it wasdemonstrated that incorporation of the sTNF-Rs into polymeric systems,such as ethylene-vinyl acetate copolymers or polylactic-glycolic acidand their subcutaneous injection, can provide systemic natural p55sTNF-Rs at high concentrations, at a constant rate for prolonged periods(more than one month) (Eliaz et al, 1966). It is thus possible thatsTNF-Rs will prove therapeutically effective in treating rheumatoidarthritis as well.

TNF, TNF-Rs and the Heart

Elevated concentrations of TNF and its soluble receptors have beendetected in sera of patients with heart failure (Levine et al, 1990).TNF may contribute to the impaired myocardial contraction in thiscondition as it was shown to produce a significant depression of myocyteshortening (Cunnion, 1990). Furthermore, whole hearts perfused withserum from animals treated with TNF 18-22 hours earlier, exhibitedsignificant impairment and decreased rate of relaxation compared tocontrols (DeMeules et al, 1992). Similar myocardial depressing effectsmay possibly be inflicted by continuous exposure of the heart to TNF,circulating in heart failure patients (Levine et al, 1990).Neutralization of the cytokine with sTNF-Rs may be useful in managingheart failure.

Inhibition of TNF

Heparin has been reported to bind TNF (Lantz et al, 1991). However, thesignificance of this observation was never examined. The effects ofheparin seem to be the exact opposite of the effects of TNF, as shown byTable I in Lantz et al.

SUMMARY OF THE INVENTION

The present invention provides for the use of heparin, and/or aderivative thereof, in the preparation of a pharmaceutical compositionfor inhibiting the bioactivity of TNF.

The present invention also provides pharmaceutical compositions forinhibiting the bioactivity of TNF.

The invention provides further a kit for the simultaneous or sequentialadministration of such a composition, comprising the active ingredientstogether with a pharmaceutically acceptable carrier, and instructionsfor use.

Heparin and low molecular weight heparins have been found to inhibit thecytokine bioactivity of TNF, particularly when acting with another TNFbinding protein. Heparin is a natural TNF binding protein, and probablycross-links TNF to its p55 TNF and p75 TNF-receptors. This inhibits thecytokine bioactivity of TNF by presumably interfering with trimerizationof the TNF receptors. The inventors raise the above theory of actionwithout being bound thereby. Thus, by administering heparin or aderivative thereof along with a soluble TNF receptor, the bioactivity ofTNF is inhibited, and the disorders caused by excess TNF can besuccessfully treated. The heparin or derivative thereof can beadministered simultaneously with the TNF receptor, either in separatecompositions or in compositions containing both heparin or a derivativethereof and at least one soluble TNF receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs showing inhibition of TNF activity by heparin(FIG. 1A) and Clexane® (FIG. 1B).

FIG. 2 is a graph showing the effect of pretreatment of cells withheparin on TNF cytotoxicity as a function of time the cells were exposedto heparin prior to TNF addition that heparin was added.

FIGS. 3A and 3B show the influence of supernatant removal on thecytotoxic effect of TNF.

FIG. 4A shows the interactions of heparin with p55 sTNF-R and TNF.

FIG. 4B shows the interactions of Clexane® with p55 sTNF-R and TNF.

FIG. 5A shows the interaction of heparin with p75 sTNF-R and TNF.

FIG. 5B shows the interactions of Clexane® with p75 sTNF-R and TNF.

FIG. 6A shows the effect of heparin added at different time points afterTNF application.

FIG. 6B shows the effect of Clexane® added at different time pointsafter TNF application.

FIG. 7 illustrates interactions between TNF, soluble TNF-receptors, andheparin or low molecular weight heparin.

FIG. 8 shows the equilibrium between TNF and its soluble receptors.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that heparin or low molecular weight heparin isable to enhance the effect of sTNF-Rs, apparently in a synergisticrather than merely an additive manner.

Heparin is a glycosaminoglycan, a highly sulfated mucopolysaccharide,consisting of a heterogeneous series of repeating disaccharide unitscomposed of D-glucuronic or L-iduronic acids in a 1,4-glycosidic linkageto glucosamine. Each of the repeating units contains two sulfate estersand one N-sulfate group. Heparin is the strongest anionically chargedorganic acid substance ever isolated from a living biological system.Heparin occurs in many different body tissues, but the lung, intestinaltract, liver, and mast cells are particularly rich in heparin. Heparinis a family of linear polymers that differ in chain length and molecularweight, and its precise complete composition is unknown.

Commercially, heparin is extracted from animal tissues, most commonlyfrom bovine lungs and the intestinal mucosa of bovine, ovine, porcine,and caprine species. In any vial of therapeutically employed heparin, awide range of molecular species, ranging from 2,000 to 25,000 daltons,are present. The potency of heparin is defined in units, where one unitis the amount of heparin that will prevent the coagulation of sheepplasma by the process of recalcification. Various extracts of heparinmay range in potency, i.e., 1 mg by weight may range in potency from80-170 units. The World Health Organization maintains referencestandards for heparin.

Heparin exhibits its inhibitory effect on the blood coagulation cascadescheme by at least two different mechanisms. First, when heparincomplexes with lysine residues of antithrombin III at high affinity in a1:1 stoichiometric manner, the serine protease inhibitory effect ofantithrombin III is enhanced several fold. Second, because of its highpolyanionic charge density, heparin is able to neutralize the effect ofpositively charged activated glycoprotein coagulant serine proteases.Heparin also induces lipoprotein lipase and histaminase degradation ofhistamine, and has antiinflammatory properties as well.

Heparin has been widely used since the mid-1940s primarily for theprophylactic prevention and treatment of thrombotic diseases such asdeep vein thrombosis, pulmonary emboli, and myocardial infarction.Another principal use of heparin is to prevent blood coagulation inextra-corporeal systems, thus making possible renal dialysis; cardiacbypass surgery; cardiac, pulmonary, hepatic, and renal transplantation;extra-corporeal pulmonary bypass oxygenation; and extra-corporealcirculatory membrane ultrafiltration. Low doses of low molecular weightheparin are used for the prophylactic prevention of intravascularthrombus formation. Heparin fragments, peptides, and syntheticallyprepared peptides have also been used.

In animal models, heparin has been shown to reduce the ability ofautoimmune T lymphocytes to reach their target organ (Lider et al,1990). Heparin has also been shown to suppress experimental autoimmunediseases in rats and to prolong the allograft survival in a model ofskin transplantation in mice, when used in low doses of about 5micrograms for mice and 20 micrograms for rats, injected once a day(Lider et al, 1989).

The mechanisms behind the observed effects of heparin are believed toinvolve inhibition of release by T lymphocytes of the enzyme(s)necessary for penetration of the vessel wall, primarily the enzymeheparanase that specifically attacks the glycosaminoglycan moiety of thesub-endothelial extracellular matrix that lines blood vessels (Naparsteket al, 1984). Expression of the heparanase enzyme is associated with theability of autoimmune T lymphocytes to penetrate blood vessel walls andto attack the brain in the model disease experimental autoimmuneencephalomyelitis.

Low molecular weight heparins, with an average molecular weight of3000-6000, such as, for example, the low molecular weight heparinsdisclosed in European Patent EP 0014184, are derived from heparin. Somelow molecular weight heparins are commercially available under differenttrade names, such Fragmin®, cf. U.S. Pat. No. 4,303,651, Fraxiparin®,Fraxiparine®, U.S. Pat. Nos. 4,486,420 and 4,692,435, Lovenox®, EuropeanPatent 40144, and Clexane®, U.S. Pat. No. 3,948,917.

Low molecular weight heparins can be produced in several different ways:enrichment by fractionalization by ethanol and/or molecular sieving,e.g., gel filtration or membrane filtration of the low molecular weightheparin present in standard heparin and controlled chemical (by nitrousacid, wbw-elimination, or periodiate oxidation) or enzymatic (byheparinase) depolymerization. The conditions for depolymerization can becarefully controlled to yield products of the desired molecular weights.Nitrous acid depolymerization is commonly used. Also, the benzylic esterof heparin can be depolymerized by wbw-elimination, which yields thesame type of fragments as enzymatic depolymerization using heparinases.Low molecular weight heparin with low anticoagulant activity whichretains the basic chemical structure of heparin can be prepared bydepolymerization using periodate oxidation or by removing theantithrombin-binding fraction of low molecular weight heparin, preparedby other methods, using immobilized antithrombin for adsorption.

Fragmin® is a low molecular weight heparin with average molecular weightwithin the range of 4000-6000 dalton, produced by controlled nitrousacid depolymerization of sodium heparin from porcine intestinal mucosa.It is manufactured by Kabi Phannacia, Sweden, under the name Fragmin®for use as an antithrombotic agent as saline solutions for injection insingle dose syringes of 2500 IU/0.2 ml and 5000 IU/0.2 ml, correspondingto about 16 mg and 32 mg., respectively.

Fraxiparin® and Fraxiparine® are low molecular weight heparins withaverage molecular weight of approximately 4500 dalton, produced byfractionation or controlled nitrous acid depolymerization, respectively,of calcium heparin from porcine intestinal mucosa. These low molecularweight heparins are manufactured by Sanofi (Choay Laboratories) for useas an antithrombotic agent in single doses comprising about 36 mg.,corresponding to 3075 IU/0.3 ml water.

Lovenox®(Enoxaprain/e), a low molecular weight heparin fragment producedby depolymerization of sodium heparin from porcine intestinal mucosausing wbw-elimination, is manufactured by Pharmuka SF, France, anddistributed by Rhone-Poulenc under the names Clexane® and Lovenox® foruse as antithrombotic agents in single dose syringes comprising 20mg/0.2 ml and 40 mg/0.4 ml water.

Low molecular weight heparins, produced by fractionalization orcontrolled depolymerization of heparins, show improved antithromboticperformance but also different pharmacokinetic properties as compared toheparin. The half-life is doubled and the bioavailbailty is higher withrespect to their anticoagulant effect following subcutaneous injection(Bratt et al, 1985; Bone et al, 1987).

The properties of the low molecular weight heparins described above area common feature to all low molecular weight heparins, regardless of themanufacturing process, the structural differences (created bydepolymerization or those dependent on variation in the heparin used asraw material) or the anticoagulant activity, provided the low molecularweight heparin used is capable of inhibiting TNF secretion in vitro byresting T cells and/or macrophages in response to activation by contactwith specific antigens, mitogens, disrupted extracellular matrix or itsprotein components, such as fibronectin or laminin.

To test the effectiveness of inhibition of activity of TNF (see Examples1 and 2), WISH cells were seeded at a concentration of 30,000 cells perwell in 100 μl medium. Sixteen hours later, when the cells had reachedabout 90% confluence, TNF, receptors, heparin, or Clexane® were added tothe wells. The final concentrations added to the respective wells are asfollows:

TNF 0.5 ng/ml

Heparin, 1 unit/ml

Clexane®, 1 unit/ml

TBPI (p55 TNF-receptor), 5 ng/ml

TBPII (p75 TNF-receptor), 10 ng/ml

The different combinations were mixed in an Eppendorf tube reaching afinal volume of 300 μl. Then, 50 μl were added to the respective well.If incubation was required, it was conducted in the Eppendorf tube for30 minutes at 37° C., in a mixture or separately. After each of thedifferent combinations were added, 50 μl cycloheximide was added to eachwell. Sixteen hours later, the cell supernatants were discarded andneutral red dye was added for one hour at 37° C. The dye was extractedfrom surviving cells with a Sorensen's solution and the results wereread in an ELISA reader.

The addition of one unit/ml of heparin to 0.5 ng/ml of TNF was found tohave about 25% of the bioactivity of TNF (i.e., a reduction ofcytotoxicity from 49% to 37%), as shown in FIG. 1A. Clexane® had a verysimilar effect, as shown in FIG. 1B. Thus, both heparin and Clexane®, alow molecular weight heparin, inhibited the toxicity of TNF.

Heparin is believed to interfere with the cellular binding of TNF. Asdetailed in Example 3, WISH cells were pretreated with heparin between 0and 30 minutes prior to TNF addition. This pretreatment substantiallyinhibited TNF cytotoxicity, as shown in FIG. 2. The heparin inhibitoryeffect was expressed immediately (at time 0), suggesting that it is notdue to a metabolic effect which induces cell resistance to TNF.

The possible explanations of this phenomenon are:

(1) Heparin may bind to TNF receptors (cell surface or solublereceptors), interfering with the binding of TNF to its receptors.

(2) Heparin does not affect TNF receptors. It may complex with TNF themoment it is applied, and/or may interfere with ligand binding tocell-associated receptors and prevent TNF from inducing receptortrimerization, which is a prerequisite for signal transduction.

As seen in Example 4, elimination of heparin or low molecular weightheparin from supernatant instantaneously eliminates their protectiveeffect against TNF cytotoxicity. Thus, the presence of heparin or a lowmolecular weight heparin is required for inhibiting TNF activity. Thepolysaccharides do not induce a metabolic state of resistance in cellspretreated with these polysaccharides. There is no affinity betweenheparin or low molecular weight heparin and cell membrane elements, suchas TNF receptors, since simple mechanical washing practically removes itand abolished the TNF-inhibitory effects.

It may, therefore, be assumed that, since the “cellular effect” ofheparin is removable, it is likely that the TNF-inhibitory effect ofheparin is due to its adherence to TNF, preventing its association withthe cell membrane TNF-Rs or interfering with it.

As detailed in Example 5, examination of the binding of heparin orClexane® to the soluble TNF receptors or to the complex TNF/TNF-Rrevealed the following, with reference to FIGS. 4 and 5:

(1) Preincubation of TNF with heparin or Clexane® for 30 minutes, andtheir application to the WISH cells, further potentiated their TNFinhibitory effect compared to their applications without preincubation(compare FIGS. 4A, 4B, 5A, 5B, columns 3 vs. 6). There appears to be aninterference phenomenon. Following preincubation with heparin or lowmolecular weight heparin, more TNF is bound to the polysaccharide whichmay interfere with TNF binding to its cell associated receptors. Analternative explanation is that heparin may promote dissociation of theactive trimer into inactive monomers.

(2) While p55 sTNF-R or p75 sTNF-R alone could inhibit about 8-15% ofthe TNF bioactivity, the addition of either heparin or Clexane® to TNFand either receptor potentiated the inhibition by the receptors 3-4times (60% inhibition). Compare FIGS. 4 and 5, columns 2, 4 and 5.

Thus, heparin or low molecular weight heparin may augment the solubleTNF-R binding to TNF, potentiating three to four times the neutralizingeffect of both p55 sTNF-R and p75 sTNF-R. It appears that thepolysaccharide cross-links TNF to its receptors. Although the conclusionthat heparin prevents TNF binding to its cell associated receptors andthe conclusion that heparin augments this binding to the solublereceptors seems contradictory and paradoxical, both conditions result inan inhibition of TNF bioactivity and may coexist.

(3) Simple preincubation of p55 sTNF-R with TNF, unlike itspreincubation with p75 sTNF-R, resulted in superior TNF inhibition, asshown in FIGS. 4A and SA, and 4B and 5B, comparing columns 4 and 7. Thismay be related to the “ligand passing” effect of p75 sTNF-R.

(4) Thirty-minute preincubation of TNF with p55 sTNF-R/p75 sTNF-R andheparin/Clexane® resulted in almost the same TNF inhibition observedwhen the three components were applied over cells without preincubation(cf. FIGS. 4 and 5, comparing columns 5 to 8). From this, one canconclude that the interaction among TNF, soluble receptor, and heparinis instantaneous, unlike the interaction of TNF-heparin, which isaugmented with time, as shown above. This suggests that the naturaltendency of TNF to bind instantaneously to its receptor may be followedby quick cross-linking of the complex formed by heparin/Clexane®.

(5) TNF was preincubated with heparin/Clexane® for thirty minutes andjust prior to their application to cells, the p55 sTNF-R or p75 sTNF-R,respectively, was added. The observed TNF cytotoxicity was highercompared to the simultaneous preincubation of the three components, asshown in FIGS. 4 and 5, comparing columns 8 to 9. One explanation isthat, during the TNF+heparin/Clexane® incubation, the polysaccharidecomplexed with TNF, interfering with its binding to the solublereceptors upon their later addition. Since free TNF, heparin, and theircomplex were at equilibrium, elimination of free TNF by addition ofsoluble receptors resulted in dissociation of TNF/polysaccharidecomplexes in order to regain the equilibrium, and free TNF had an equalchance to bind the soluble receptors of the cell receptors and activatethem. Further support for heparin/Clexane®'s interference with TNFbinding to its receptor was gained when comparing column 9 to column 5in FIGS. 4 and 5.

(6) Thirty-minute preincubation of p55 sTNF-R or p75 sTNF-R with eitherheparin/Clexane® and addition of TNF just before application to thecells, as shown in FIGS. 4A and 5A, and 4B and 5B, resulted in TNFinhibition identical to that obtained if the three components were addedsimultaneously to cells (column 5) or after their joint preincubationfor thirty minutes (column 8). From this it can be concluded that thepolysaccharide does not interfere with TNF-receptor binding to TNF, andhas no affinity for the “bare” TNF receptor. However, heparin/Clexane®has a strong affinity for the TNF/TNF-receptor complex, which it avidlycross-links. Thus, since heparin/Clexane® has no affinity for the “bare”soluble TNF receptors, the ease of washing of the “cellular effect” ofheparin/Clexane®, shown in FIG. 2, is consistent with the conclusionthat the heparin/Clexane® has no affinity for the “bare” cell associatedreceptors as well.

(7) The inhibition of TNF after its preincubation with its p55 sTNF-Rand addition of heparin/Clexane® just before application to cells wasbetter (FIGS. 4A and 4B, column 11) than the inhibition obtained afterTNF preincubation with p55 sTNF-R only, as shown by a comparison ofFIGS. 4A and 4B, columns 7 and 11. This suggests that heparin/Clexane®further facilitates the inhibition of TNF by p55 sTNF-R, probably bytheir cross-linking. It should be noted that following heparin/Clexane®addition, the TNF inhibition by p75 sTNF-R was better (20-25%) than theinhibition of p55 sTNF-R (15%). This can be seen by comparing FIGS. 4Aand 4B, columns 7 and 11, with FIGS. 5A and 5B, columns 7 and 11.Heparin/Clexane® potentiates TNF binding to its p55 sTNF-Rr and p75sTNF-R. The greater TNF inhibition by p75 sTNF-R in the presence of thepolysaccharide may be related to prevention of “ligand passing” by p75sTNF-R when heparin/Clexane® cross-links it to TNF.

Heparin/Clexane® potentiates binding of TNF to its soluble receptors,thus augmenting their TNF inhibitory effect. However, one would expectthat a similar enhanced binding to cell associated receptors, shown inFIGS. 4 and 5, comparing column 2 to column 3, would result in enhancedTNF cytotoxicity. In practice, though, TNF's cytotoxicity was inhibited.

One theoretical explanation for this apparent paradox is thatheparin/Clexane® promotes cross-linking of the bioactive TNF trimer toonly one or two TNF receptors, thus interfering with the binding of thethird receptor to it. Promoting such binding to soluble TNF receptorsneutralizes TNF bioactivity. Obviously, potentiating TNF binding to onlyone or two cell surface receptors, while interfering with the finalreceptor aggregation into trimers, explains the above paradox, as signaltransduction is best elicited upon aggregation of three cell surfacereceptors. Following cross-linking of one cell surface receptor to TNFby heparin/Clexane®, the polysaccharide may become interposed in a waythat may prevent further cell surface TNF-receptor trimerization. On theother hand, if the TNF already induced receptor trimerization, heparincannot cross-link this complex or interfere with its function. This maybe the key to TNF inhibition by this polysaccharide.

In support of the above explanation, it was noted that there was aparadoxical effect in the experiments. In experiments where loweramounts of soluble receptors were used, characterized by a minimal (lessthan 10% TNF inhibition), the potentiating effect of heparin/Clexane®was maximal (more than 60% inhibition). In experiments in which thereceptors exerted a 50% inhibition, heparin/Clexane® had a marginalpotentiating effect.

It appears that with very low amounts of soluble receptors, most TNFtrimers can produce complexes with only one soluble receptor. Thesecomplexes are the probable target of heparin/Clexane® cross-linking,resulting in a remarkable TNF inhibition. If the amount of the solublereceptors is higher, two or more soluble receptors may bind to TNF,preventing their effective cross-linking of such complexes by thepolysaccharide. It remains to be demonstrated that these complexes arenot stabilized by heparin/Clexane® to the same extent as are complexesof TNF and monomeric soluble receptor.

Strong supporting evidence that the heparin/Clexane® cross-linking maybe limited to complexes of TNF with one or, at most, two receptors comesfrom comparison of columns 5 to 11 of FIGS. 4 and 5. If an equilibriumwas attained between TNF+p55 sTNF-R with formation of TNF complexes withone, two or three receptors, and then heparin/Clexane® was added (column11), the TNF cytotoxicity was enhanced compared to simultaneous additionof the TNF, its receptors and heparin over cells (column 5). A possibleexplanation is that, in the latter situation, TNF could initially bindone single receptor. This complex would be immediately cross-linked byheparin/Clexane®, preventing TNF from binding a second or thirdreceptor.

Additional supporting evidence can be found in the experiment todetermine if heparin/Clexane® can inhibit the bioactivity of TNF ifheparin/Clexane® is added after TNF application. If heparin/Clexane® wasapplied at different time periods after TNF application, its inhibitoryactivity was still significant after 15 minutes, and marginallypersistent after one hour. Surprisingly, in two experiments, theinhibitory activity of heparin/Clexane® was about 7-15% at time 0,increased to 25% if applied 5-15 minutes after TNF, and decreased to 10%at one hour (FIG. 6).

One explanation for this paradoxical increase if heparin/Clexane® isadded 5-15 minutes after TNF is that heparin/Clexane® binds avidly onlyone monomer-receptor to a trimer TNF. This binding interferes withfurther receptor trimerization, which is known to be followed by signaltransduction.

One can visualize the binding of TNF to its receptors as a processduring which, at the beginning, TNF is bound to one receptor, with timeis bound to the second receptor, and with additional time to the thirdreceptor. The fact that heparin/Clexane® can still inhibit TNF even onehour after its application suggests that it may interfere at this latestage with trimerization of the few last receptors. TNF molecules may becombined at this later stage with one receptor or two, andheparin/Clexane® interferes with the binding of the third, which wouldotherwise induce signal transduction.

This mechanism may explain the paradox that addition of heparin/Clexane®fifteen minutes following TNF application results in a better inhibitionof TNF than when applied simultaneously. At time 0, heparin/Clexane®binds part of the TNF and may slightly interfere with its binding to thecell receptors, as noted above. If low concentrations of TNF are appliedseveral minutes before heparin/Clexane®, TNF has the opportunity to bindto its receptors undisturbed, and application of the heparin now willcross-link optionally the TNF bound to receptor monomers.

It can thus be seen that treating a patient with heparin and/or a lowmolecular weight heparin can inhibit the bioactivity of TNF. The effectof heparin or derivatives thereof can be potentiated by administeringp55 sTNF-R or p75 sTNF-R in combination with the heparin or derivativethereof. The heparin and soluble receptors can be administeredsimultaneously, or over approximately a 15-30 minute interval.Alternatively, the heparin or derivative is administered, andapproximately 15-60 minutes later p55 sTNF-R or p75 sTNF-R isadministered.

While heparin per se can be administered, low molecular weight heparin,produced by fractionation or controlled depolymerization of heparins,has improved antithrombotic performance, as well as differentpharmacokinetic properties, as compared to heparin. The half-life of thelow molecular weight heparins is doubled. However, even though Bratt etal (1985) found that their bioavailability is higher with respect totheir anticoagulant effect after subcutaneous injection, it should benoted from FIGS. 4 and 5 that heparin and Clexane® interact with p55sTNF-R, p75 sTNF-R, and TNF approximately the same. Thus, any lowmolecular weight heparin can be used in place of heparin for the purposeof the present invention.

The low molecular weight heparins that can preferably be used in thepresent invention include Clexane®, as described above, as well asFragmin®, a low molecular weight heparin with average molecular weightwithin the range of 4000-6000 daltons, produced by controlled nitrousacid depolymerization of sodium heparin from porcine intestinal mucosa,manufactured by Kabi Pharmacia, Sweden. Also useful are Fraxiparin® andFraxiparine®, low molecular weight heparins with average molecularweight of approximately 4500 daltons, produced by fractionation orcontrolled nitrous acid depolymerization, respectively, or calciumheparin from porcine intestinal mucous manufactured by Sanofi (ChoayLaboratories).

For purposes of the present invention, heparin per se can be used,either alone or in combination with a low molecular weight heparin,regardless of the manufacturing process, the structural differences(created by depolymerization or those dependent on variation in theheparin used as raw material), or the anticoagulant activity.Alternatively, a low molecular weight heparin can be used alone.

The disorders that can be treated by inhibiting TNF activity accordingto the present invention are all disorders related to the presence ofTNF and which respond to inhibition of the bioactivity of TNF. Amongthese disorders are atherosclerosis and vasculitis and pathologicalprocesses related thereto; autoimmune diseases, such as rheumatoidarthritis, diabetes mellitus type I; allergies; graft rejection; acuteand chronic inflammatory diseases, such as uveitis and bowelinflammation; anorexia nervosa; hemorrhagic shock caused by septicemia;and opportunistic infections in AIDS-compromised individuals.

Heparin or a low molecular weight lieparin, or mixtures thereof, isincorporated into pharmaceutical compositions, for example, as watersolutions, possibly comprising sodium chloride, stabilizers, and othersuitable non-active ingredients. The preferred method of administrationis by injection, subcutaneous or intravenous, but any other suitablemode of administration is encompassed by the invention.

The soluble TNF receptors, p55 sTNF-R and p75 sTNF-R, are likewiseincorporated into pharmaceutical compositions, either alone or incombination with heparin and derivatives thereof. The amounts of heparinor derivatives thereof administered depend upon the mode ofadministration. If a slow release preparation is administered, theamounts administered will be much lower than if administeredintramuscularly or intravenously.

Pharmaceutical compositions for administration according to the presentinvention can comprise at least one heparin or derivative thereof and atleast one soluble TNF receptor, either separately or together, in apharmaceutically acceptable form optionally combined with apharmaceutically acceptable carrier. These compositions can beadministered by any means that achieve their intended purposes. Amountsand regimens for the administration of a composition according to thepresent invention can be determined readily by those with ordinary skillin the art of treating disorders related to excessive bioactivity ofTNF.

For example, administration can be by parenteral, such as subcutaneous,intravenous, intramuscular, intraperitoneal, transdennal, or buccalroutes. Alternatively or concurrently, administration can be by the oralroute. The dosage administered depends upon the age, health and weightof the recipient, type of previous or concurrent treatment, if any,frequency of the treatment, and the nature of the effect desired.

Compositions within the scope of this invention include all compositioncomprising at least one heparin or derivative administered incombination with at least one soluble TNF receptor in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. Typical dosages comprise about 0.1 toabout 100 mg/kg body weight.

The following non-limiting examples will help to explain the presentinvention.

EXAMPLE 1 Inhibition of TNF Activity by Heparin

WISH cells were seeded at a concentration of 30,000 cells/well in 100 μlmedium. Sixteen hours later, either medium (control), heparin, TNF or acombination of TNF plus heparin was added. The final concentrations ofeach into the respective wells were as follows: TNF 0.5 ng/ml; heparin 1unit/ml. After the various additions, cyclohexamide was added to eachwell (50 μl) for a final concentration of 25 μg/ml in the well. Sixteenhours later, the cell supernatants were discarded and neutral red dyewas added for one hour. Following the one hour incubation, the dye wasextracted with a Sorensen's solution, and the results were read in anELISA reader. The results directly correlate with percent of cellkilling. The results are shown in FIG. 1A. It can be seen that theaddition of TNF inhibits about 25% of the bioactivity of TNF (reductionof cytotoxicity from 49% to 37%).

EXAMPLE 2 Inhibition of TNF Activity by Clexane®

The same procedure as in Example 1 was repeated except that Clexane® wassubstituted for heparin. The results are shown in FIG. 1B. It can beseen that substantially similar inhibitory effects are obtained.

EXAMPLE 3 Effect of Pre-Treatment with Heparin on TNF Cytotoxicity

The same procedure as in Example 1 was repeated except that the WISHcells were treated with heparin between 0-30 minutes before addition ofthe TNF. The results are shown in FIG. 2. It can be seen thatsubstantially identical inhibitory effects are obtained throughout thetimeline.

EXAMPLE 4 Effect of Heparin Removal by Washing

WISH cells were seeded at a concentration of 30,000 cells per well in100 μl medium. Sixteen hours after seeding of the cells either heparinor Clexane® was added to respective wells, while medium alone was addedto control wells. The heparin or Clexane® was added to a finalconcentration of 1 unit/ml. Six hours later, part of the wellspretreated with either heparin or Clexane® or with medium only werewashed three times with fresh medium, and TNF was added to a finalconcentration of 0.5 ng/ml. The results are shown in FIG. 3A for heparinand FIG. 3B for Clexane®. It can be seen that heparin/Clexane®pretreatment reduced the TNF cytotoxicity by 33% as expected (from 60%to 40%) (compare columns 2 to 4 in FIGS. 3A and 3B). However, simplewashing of the cells treated with medium also resulted in a 25%reduction in the cell susceptibility to TNF. Cells pretreated withheparin or Clexane®, whose supernatants were washed before TNF addition,had an identical killing by TNF (compare columns 3 to 5 of FIGS. 3A and3B). Thus, elimination of heparin or Clexane® from the supernatantseliminates instanteously their protective effect against TNFcytotoxicity.

Removal of the supernatants, washing the cells and addition of newmedium increases the cell resistance to TNF by 20%. It is possible thatthe removed supernatants contain a factor that facilitates TNFcytotoxicity and its elimination reduces the effect of TNF. Anotherpossibility is that, following removal of the supernatants, there israpid shedding of the cell surface TNF receptors, as previously found(Aderka, in press), which may induce some transient desensitization toTNF.

EXAMPLE 5 Effect of Heparin/Clexane® on TNF Receptors

WISH cells were seeded at a concentration of 30.000 cells/well in 100 μlmedium. Sixteen hours later, either medium alone (control), or sTNF-R(either p55 sTNF-R (FIGS. 4A and 4B) or p75 sTNF-R (FIGS. 5A and 5B)),and either heparin (FIGS. 4A and 5A) or Clexane® (FIGS. 4B and 5B) wereadded. The final concentrations of each in the respective wells were asfollows: TNF 0.5 ng/ml; heparin 1 unit/ml; Clexane® 1 unit/ml; TBP-I(p55 TNF-receptor) 5 ng/ml; TBP-II (p75 TNF-receptor) 10 ng/ml.

Different combinations of the ingredients were mixed in an Eppendorftube reaching a final volume of 300 μl. 50 μl were then added to eachrespective well. If an incubation was required, it was done in anEppendorf tube as a mixture or separately for 30 minutes at 37° C.

After addition of the different combinations, cyclohexamide was added toeach well (50 μl) for a final concentration of 25 μg/ml. Sixteen hourslater, the cell supernatants were discarded and neutral red dye wasadded for one hour. Following the one hour incubation, the dye wasextracted with a Sorensen's solution and the results were read in anELISA reader.

The results are shown in FIGS. 4A, 4B, 5A and 5B. In each of thesefigures, the first bar of the graph represents a control in which theEppendorf tube included 300 pi of medium only. In the second bar ofeach, the Eppendorf tube included 2.1 μl of TNF at a concentration of 2ng/ml plus 20 μl of medium.

In bar 3 of each figure, 1.8 units of either heparin or Clexane® (10 μl)and 10 μl of medium were added to the Eppendorf tube along with theaddition of 280 μl of TNF at a concentration of 2.1 ng/ml. The mixturewas then immediately added to the WISH cell wells.

With respect to the fourth bar, 6 ng p55 sTNF-R (10 μl) were added to 10μl of medium and the same amount of TNF discussed above, immediatelyprior to addition of to the WISH cell wells. In FIGS. 5A and 5B, doublethe amount of p75 sTNF-R (12 ng/10 μl) was used in place of p55 sTNF-R.

With respect bar 5, 6 ng of p55 sTNF-R or 12 μg of p75 sTNF-R (10 μl),1.2 units of either heparin or Clexane® (10 μl), and 280 μl TNF at 2.1ng/ml were added to the Eppendorf tube. The mixture was then immediatelyadded to the WISH cell wells.

Bar 6 involves the same materials as discussed above for bar 3, exceptthat the TNF and either heparin or Clexane® were added simultaneously tothe Eppendorf tube and then incubated together for 30 minutes prior tobeing added to the WISH cell wells. Similarly, bar 7 is the same asdescribed above for bar 4, except that either the p55 sTNF-R or p75sTNF-R was mixed with the TNF and incubated together for 30 minutes at37° C. before being added to the WISH cell wells. The experiment of bar8 is the same as that described above for bar 5, except that the TNF,either the p55 sTNF-R or the p75 sTNF-R, and either the heparin orClexane® were mixed together and incubated for 30 minutes before beingadded to the WISH cell wells.

For bar 9, the TNF and either heparin or Clexane® were pre-incubatedtogether for 30 minutes before addition of either the p55 sTNF-R or p75sTNF-R which were pre-incubated separately and then immediate additionto the WISH cell wells. For bar 10, either the p55 sTNF-R or the p75sTNF-R and either the heparin or Clexane® were pre-incubated togetherfor 30 minutes before addition of the pre-incubated TNF and thenimmediate addition to the WISH cell wells. For bar 11, the TNF andeither p55 sTNF-R or p75 sTNF-R were incubated together for 30 minutesbefore addition of either the pre-incubated heparin or Clexane® and thenimmediate addition to the WISH cell wells.

As can be seen from a comparison of the various bars of FIGS. 4A, 4B, 5Aand 5B, pre-incubation of TNF with heparin/Clexane® for 30 minutes andtheir application to the WISH cells further potentiated their TNFinhibitory effect compared to their application without pre-incubation(comparing bars 3 and 6 of each). Furthermore, while p55 sTNF-R or p75sTNF-R alone could inhibit about 8-15% of the TNF bioactivity, additionof either heparin or Clexane® to TNF and either receptor potentiated theinhibition by the receptors three to four times (60% inhibition)(comparing columns 2, 4 and 5 of the various figures).

Kits for the simultaneous or sequential administration of heparin and/ora derivative thereof, and a soluble TNF receptor, are prepared in aconventional manner. Typically, such a kit will comprise, e.g. anampoule of each of the active ingredients in a pharmaceuticallyacceptable carrier, a syringe, and written instructions for thesimultaneous or sequential administration. For example, if simultaneousadministration is desired, the contents of the ampoules may be mixedprior to injection in either a suitable vessel, or in the syringeitself.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention. Thusthe expressions “means to . . . ” and “means for . . . ”, or any methodstep language, as may be found in the specification above and/or in theclaims below, followed by a functional statement, are intended to defineand cover whatever structural, physical, chemical or electrical elementor structure, or whatever method step, which may now or in the futureexist which carries out the recited function, whether or not preciselyequivalent to the embodiment or embodiments disclosed in thespecification above, i.e., other means or steps for carrying out thesame function can be used; and it is intended that such expressions begiven their broadest interpretation.

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What is claimed is:
 1. A composition for inhibiting the bioactivity ofTNF, comprising an effective amount of heparin or a low molecular weightderivative thereof, or a mixture of heparin and a low molecular weightderivative thereof, and at least one sTNF-R.
 2. A composition accordingto claim 1, further comprising a pharmaceutically acceptable carrier. 3.A composition according to claim 1, wherein the sTNF-R is selected fromthe group consisting of p55 sTNF-R and p75 sTNF-R.
 4. A compositionaccording to claim 1, wherein the heparin or a low molecular weightderivative thereof is a low molecular weight heparin.
 5. A compositionaccording to claim 4, wherein the low molecular weight heparin has amolecular weight between 2500 and 6500 daltons.
 6. A kit for thesimultaneous or sequential administration of a composition according toclaim 1, comprising the active ingredients together with apharmaceutically acceptable carrier, and instructions for use.
 7. Acomposition comprising heparin and/or a low molecular weight derivativethereof and a sTNF-R.
 8. In a method for inhibiting the activity of TNFin a subject comprising treating said subject with an effective amountof sTNF-R, the improvement whereby the effect of sTNF-R is potentiatedcomprising treating said subject with a combination of said sTNF-R withan amount of heparin or a low molecular weight derivative or a mixtureof heparin and a low molecular weight derivative thereof, said amountbeing effective to potentiated the effect of said sTNF-R.
 9. A method inaccordance with claim 8, wherein said sTNF-R is selected from the groupconsisting of p55 sTNF-R and p75 sTNF-R.
 10. A method in accordance withclaim 9, wherein said heparin or low molecular weight derivative thereofis administered simultaneously with said sTNF-R.
 11. A method inaccordance with claim 9, wherein said heparin or low molecular weightderivative thereof is administered within one hour after administrationof said sTNF-R.
 12. A method in accordance with claim 8, wherein saidheparin or low molecular weight derivative thereof is a low molecularweight heparin.
 13. A method in accordance with claim 12, wherein saidlow molecular weight heparin has a molecular weight between 2500 and6500 daltons.